The molecular response to environmental and physiological stress alerts cells to imminent danger and serves to protect the genetic and biosynthetic apparatus from sustaining potentially lethal proteotoxic damage. The heat shock response adjusts the levels of molecular chaperones to prevent the appearance and accumulation of aggregation-prone polypeptides during cell stress. At the center of this regulatory network, in larger eukaryotes, is a family of heat shock transcription factors (HSFs). Under conditions of normal cell growth, HSFs are repressed. In response to stress, HSFs are rapidly activated to induce the transcription of heat shock genes. The levels of chaperones are carefully balanced to reflect the levels of misfolded proteins and heat shock factor activity, such that the heat shock response is autoregulated and attenuates during recovery. This is a proposal for continued funding to: (1) Order the events associated with activation and attenuation of the heat shock response. The experiments would elucidate the process of intramolecular negative regulation of both the DNA binding and transactivation domains, the role of the molecular chaperone Hsp90 in maintenance of the inert monomer, the role of Hsp70/Hdj1 in transcriptional repression, the function of HSBP1 in the negative regulation of the heat shock response, and the characterization of other trans-regulators of HSF1. (2) Characterize the function of HSF1 stress granules in the cell biology of the heat shock response. Experiments are proposed to identify the targeting and retention signals involved in localization of HSF1 to stress granules, to identify the stress-regulated nuclear transport mechanism, to identify the chromosomal location of HSF1 granules, and to establish the role of HSF1 stress granules in the heat shock response. (3) Examine how different members of the family of heat shock factors coordinate their activities. Experiments are proposed to understand how the activities of HSF1 and HSF2 are coordinated under normal conditions of cell growth and stress and to establish the regulatory link between HSF2 lability and control by general protein degradation and the ubiquitin-dependent proteasome. (4) Characterize the C. elegans heat shock response to understand how stress responses are regulated in a multicellular differentiating organism. Dr. Morimoto will focus on tissue specific, developmental, and aging signals which modulate the heat shock response in C. elegans; the function of C. elegans HSF and trans-regulators, including heat shock proteins and HSB-1; and the relationship of the heat shock response to diseases of protein misfolding and protein aggregation, including Huntington's Disease and ALS. Proteins containing beta-hairpin stacked sheets will be expressed in C. elegans and human tissue culture cells (neuronal and non-neuronal), to understand the molecular basis of the "stress signaling".